The long-term objectives of this research proposal are to acquire a thorough understanding of the mechanisms of action and of resistance to Taxol, an antitumor agent that is known to be efficacious in the treatment of human cancer. Knowledge gained from the studies described in this proposal will improve our ability to design Taxol analogs with a better therapeutic index, to develop strategies to reverse or overcome drug resistance and to understand the synergy between Taxol and discodermolide that we have reported in tissue culture cells. Our understanding of Taxol will be applied to other natural products such as the epothilones and discodermolide whose mechanisms of action and resistance have major similarities, but are not identical, to those of Taxol. The specific objectives of the research proposal are to: 1. Define the binding site(s) for epothilone B and discodermolide in the microtubule and determine the relationship between these binding sites and that of Taxol. Drug analogs with radiolabeled photoreactive substituents at defined positions in the molecules will be used to determine the sites of interaction between the drug and its target, the microtubule. 2. Investigate the basis of resistance to Taxol, epothilone and discodermolide in drug resistant cell lines that do not express P-glycoprotein. Tubulin harboring mutations will be analyzed for functional alterations. We will also determine if Taxol, epothilone and discodermolide bind preferentially to a single beta-tubulin isotype. These experiments will take advantage of a new mass spectrometry-based method that has been developed in our laboratory for the analysis of the C-terminal diversity of a- and beta-tubulin isotypes. 3. Determine if the synergism between Taxol and discodermolide that has been demonstrated in four cancer cell lines in our laboratory can be attributed to specific structural/functional characteristics of discodermolide. The study will take advantage of unique discodermolide analogs in a series of in vitroand mouse experiments.